磷酸Akt(Ser473)检测试剂盒(基于细胞比色法检测)
Phospho-Akt (Ser473) Detection Kit B(Cell-based Colorimetric Assay)

Akt plays an essential role in cell survival, growth, migration, proliferation, polarity, and metabolism (lipid and glucose). Akt is activated through receptor tyrosine kinase pathways. Thr308 and Ser473 phosphorylation are necessary and sufficient for the full activation of Akt. Quantification of intracellular proteins and phosphorylation events is extremely important for biomedical research. The plate-based immunoassay has become a popular alternative method for rapid protein detection.

The ABS_Bio™ Cell-Based Phospho-Akt (Ser473) Colorimetric Detection Kit is a very rapid, convenient and sensitive assay kit enable simultaneous detection of both total and phospho-Akt (Ser473) in cells, eliminating the need for lysate and protein preparation. Cells are grown in 96-well plates and treated with the appropriate conditions, such as inhibitors or ligand stimulation. The cells are then fixed and permeabilized in the wells. This is followed by parallel incubation with two primary antibodies: a phospho-specific antibody (pAkt-Ser473) and an anti-Akt antibody, after washing away unbound antibody, HRP-conjugated secondary antibody was added, after washing step, the TMB substrate was used for color developing. Therefor the relative amount of phospho-specific and total Akt protein are determined using target-specific primary antibody and HRP conjugated secondary antibody detection agent. Following the colorimetric measurement of HRP activity, the Janus Green cell staining is used for counting cell number to normalize the well-to-well different. The kit provides reagents sufficient for 100 assays. Cell-Based ELISAs are amenable to high-throughput applications and may prove a valuable addition to kinase inhibitor screening strategies.

试剂盒组分:

Storage and Handling: Store kit at -20+4°C, protected from light. Shelf life: 6 months after receipt. Warm Reagents to room temperature before use.

应用:Measurement of Akt phosphorylation level in whole cells, Cell based screening kinase inhibitors and ligand stimulation.

反应种属:Human, Mouse, Rat

样品类型:Culture cell lines-whole cell assay

检测方法:Colorimetric cell based ELISA assay, (OD450 nm; 595 nm)

时间:3-4 hrs

存储:-20 +4°C

保质期:6 months after receipt.

运输:Icepacks

所需其他材料: 

37% formaldehyde (Molecular Biology Grade; Sigma, Catalog # F8775), refer to MSDS prior to use 3% H2O2 (Sigma, Catalog # 323381).

refer to MSDS prior to use Tissue culture clear-bottom Corning 96 well plate (Sigma, cat# CLS3628).

Multi-channel pipette for washing Cell culture incubator Orbital shaker micro plate reader